In Silico, In Vitro, and Ex Vivo Biological Activity of Some Novel Mebeverine Precursors

Irritable bowel syndrome (IBS) is a functional gastroenterological disorder with complex pathogenesis and multifaceted therapy approaches, aimed at alleviating clinical symptoms and improving the life quality of patients. Its treatment includes dietary changes and drugs from various pharmacological groups such as antidiarrheals, anticholinergics, serotonin receptor antagonists, targeting chloride ion channels, etc. The present article is focused on the synthesis and biological evaluation of some mebeverine precursors as potential antispasmodics. Methods: In silico analysis aimed at predicting the pharmacodynamic profile of the compounds was performed. Based on these predictions, ex vivo bioelectrical activity (BEA) and immunohistochemical effects of the compounds were established. A thorough biological evaluation of the compounds was conducted assessing their in vitro antimicrobial and cytotoxic activity. Results: All the newly synthesized compounds exerted drug-like properties, whereby 3-methyl-1-phenylbutan-2-amine 3 showed a significant change in BEA due to Ca2+ channel regulation, Ca2+ influx modulation, and a subsequent change in smooth muscle cell response. The immunohistochemical studies showed a good correlation with the obtained data on the BEA, defining amine 3 as a leader structure. No cytotoxicity to human malignant leukemic cell lines (LAMA-84, K-562) was observed for all tested compounds. Conclusion: Based on the experimental results, we outlined 3-methyl-1-phenylbutan-2-amine 3 as a potential effective choice for orally active long-term therapy of IBS.


Introduction
Irritable bowel syndrome (IBS) is a complex syndrome, characterized by abdominal pain and impaired defecation. The exact pathophysiology of IBS is not completely understood [1]. The symptomatic treatment is patient specific; however, antispasmodic drugs are widely used to treat abdominal cramping [2]. Mebeverine is an anticholinergic spasmolytic drug commonly used in the management of IBS for many years (Figure 1). It also acts as a calcium channel blocker, and musculotropic agent and exerts antispasmodic activity and regulatory effects on bowel function [3]. The compound is a second-generation papaverine analog endowed with direct myolytic activity by reducing the excessive contractility of smooth muscle (SM) cells [4].
are widely used to treat abdominal cramping [2]. Mebeverine is an anticholinerg molytic drug commonly used in the management of IBS for many years ( Figure 1 acts as a calcium channel blocker, and musculotropic agent and exerts antispasm tivity and regulatory effects on bowel function [3]. The compound is a second-gen papaverine analog endowed with direct myolytic activity by reducing the excess tractility of smooth muscle (SM) cells. [4]. Despite mebeverine being used to treat IBS, it does not have a statistically sig effect on IBS symptoms [5].
Recently, we described the synthesis of 3-methyl-1-phenylbutan-2-amides, t relaxant activity, and influence on cognitive functions [6]. The compounds we posely synthesized as precursors of papaverine, which belongs to the isoquinoli and isoquinolines can be obtained from phenylethylamines [7] and are known as nin (5-HT) agonists ( Figure 2). Based on their pharmacological activities, we considered the synthesis of 3-1-phenylbutan-2-amine 3 and its amides. Our choice was also driven by the st resemblance to mebeverine, which is considered a second-generation papaveri logue, and its known pharmacological application as an IBS medication. This pr us to synthesize a number of mebeverine precursors with a C-3 substituent, as w investigate their biological activity ( Figure 3). The intrinsic biological activity of compounds can be studied both in vivo and using functionally active isolated tissues. The ex vivo method is performed on tissues capable of responding to physiological stimuli and is routinely applied to potential biological action of newly synthesized experimental molecules and v medicinal agents [8]. In our particular study design, we were inclined to use SM c Despite mebeverine being used to treat IBS, it does not have a statistically significant effect on IBS symptoms [5].
Recently, we described the synthesis of 3-methyl-1-phenylbutan-2-amides, their SM relaxant activity, and influence on cognitive functions [6]. The compounds were purposely synthesized as precursors of papaverine, which belongs to the isoquinoline class and isoquinolines can be obtained from phenylethylamines [7] and are known as serotonin (5-HT) agonists ( Figure 2). acts as a calcium channel blocker, and musculotropic agent and exerts antispasmodic activity and regulatory effects on bowel function [3]. The compound is a second-generation papaverine analog endowed with direct myolytic activity by reducing the excessive contractility of smooth muscle (SM) cells. [4]. Despite mebeverine being used to treat IBS, it does not have a statistically significant effect on IBS symptoms [5].
Recently, we described the synthesis of 3-methyl-1-phenylbutan-2-amides, their SM relaxant activity, and influence on cognitive functions [6]. The compounds were purposely synthesized as precursors of papaverine, which belongs to the isoquinoline class and isoquinolines can be obtained from phenylethylamines [7] and are known as serotonin (5-HT) agonists ( Figure 2). Based on their pharmacological activities, we considered the synthesis of 3-methyl-1-phenylbutan-2-amine 3 and its amides. Our choice was also driven by the structural resemblance to mebeverine, which is considered a second-generation papaverine analogue, and its known pharmacological application as an IBS medication. This prompted us to synthesize a number of mebeverine precursors with a C-3 substituent, as well as to investigate their biological activity ( Figure 3). The intrinsic biological activity of compounds can be studied both in vivo and ex vivo using functionally active isolated tissues. The ex vivo method is performed on isolated tissues capable of responding to physiological stimuli and is routinely applied to verify a potential biological action of newly synthesized experimental molecules and validated medicinal agents [8]. In our particular study design, we were inclined to use SM cells as a Based on their pharmacological activities, we considered the synthesis of 3-methyl-1-phenylbutan-2-amine 3 and its amides. Our choice was also driven by the structural resemblance to mebeverine, which is considered a second-generation papaverine analogue, and its known pharmacological application as an IBS medication. This prompted us to synthesize a number of mebeverine precursors with a C-3 substituent, as well as to investigate their biological activity ( Figure 3). Despite mebeverine being used to treat IBS, it does not have a statistica effect on IBS symptoms [5].
Recently, we described the synthesis of 3-methyl-1-phenylbutan-2-am relaxant activity, and influence on cognitive functions [6]. The compoun posely synthesized as precursors of papaverine, which belongs to the isoq and isoquinolines can be obtained from phenylethylamines [7] and are kno nin (5-HT) agonists ( Figure 2). Based on their pharmacological activities, we considered the synthesi 1-phenylbutan-2-amine 3 and its amides. Our choice was also driven by resemblance to mebeverine, which is considered a second-generation pa logue, and its known pharmacological application as an IBS medication. T us to synthesize a number of mebeverine precursors with a C-3 substituen investigate their biological activity ( Figure 3). The intrinsic biological activity of compounds can be studied both in viv using functionally active isolated tissues. The ex vivo method is performe tissues capable of responding to physiological stimuli and is routinely appl potential biological action of newly synthesized experimental molecules medicinal agents [8]. In our particular study design, we were inclined to us The intrinsic biological activity of compounds can be studied both in vivo and ex vivo using functionally active isolated tissues. The ex vivo method is performed on isolated tissues capable of responding to physiological stimuli and is routinely applied to verify a potential biological action of newly synthesized experimental molecules and validated medicinal agents [8]. In our particular study design, we were inclined to use SM cells as a platform for ex vivo contractility evaluation because they retain the ability to develop active tension even when isolated from the body [9,10]. SM tissue is a main structural element of a number of internal organs. It is related to the motor activity of the stomach and is a complex superposition of bioelectrical (slow wave with its characteristic frequency and amplitude) and contractile activity (CA) (tone, frequency, and amplitude of spontaneous or induced muscle contractions) which can be registered isometrically in isolated tissues [11][12][13]. The bioelectrical activity (BEA) of SM isolated from different segments of the gastrointestinal tract (GIT) varies. In the absence of stimuli of a neuronal nature or endogenous substances, the value of their membrane potential constantly fluctuates. These cyclic changes of depolarization and repolarization are known as slow waves [14]. Slow waves consist of a depolarization, a plateau phase, and a repolarization phase typical for the respective area. It is considered that their frequency is consistent with the maximum possibility of SM shortening, as well as with the physiological purpose of the region. The frequency of the slow wave is relatively constant and typical for each segment of the GIT having 3-10 waves/min [15]. Unlike frequency, the amplitude and duration of slow waves can change under the influence of various humoral factors, as well as in response to drugs and other biologically active substances. Local SM reactivity can also be modulated by neuronal signals. Neurotransmitters change input impedance and activate conductance that generates rhythmically alternating currents in and out of the Cajal cells [16]. Thus, the electrical and mechanical behavior of SM from the GIT, including the stomach, are a composite of the individual electrical activities of pacemaker cells, GM cells, and enteric motor neurons, as well as the quality of coupling between them [17].
The 5-HT 3 receptor, on the other hand, belongs to the Cys-loop superfamily of ligandgated ion channels. This ion channel is cation selective for sodium (Na + ), potassium (K + ), and calcium (Ca 2+ ) ions, and mediates neuronal depolarization and excitation in the central and peripheral nervous systems [18]. Binding of the neurotransmitter 5-hydroxytryptamine (serotonin) to 5-HT 3 receptor opens the ion channel, which in turn leads to an excitatory response in neurons. The rapidly activating desensitizing inward current is carried primarily by sodium and potassium ions [19]. The 5-HT 3 receptors are similar in structure to the nicotinic acetylcholine receptor and are also engaged in the regulation of intestinal motility, secretion, and peristalsis in the enteric nervous system and the transmission of information in the GIT [20].
The quest for new therapeutic approaches for IBS to modulate known pharmacological targets or validate new molecules with an alternative mechanism of action is still a priority. Biofilm-related infections, for example, are extremely challenging to treat and pose major concerns for public health. Biofilm formation is one of the major causes of therapeutic failure [21]. Therefore, in addition to the ex vivo spasmolytic activity of our experimental compounds, we sought to determine their in vitro antimicrobial and cytotoxic effects, using mebeverine as a reference drug.

Chemicals
All solvents and reagents were purchased from Merck (Merck Bulgaria EAD). Melting points were determined on a Boetius hot stage apparatus and are uncorrected. All the compounds were characterized by 1 H NMR, 13 CNMR, IR, and HRESIMS. The purity of these compounds was determined by TLC using several solvent systems of different polarity. TLC was carried out on precoated 0.2 mm Fluka silica gel 60 plates (Merck KGaA, Darmstadt, Germany), using chloroform: diethyl ether: n-hexane = 6:3:1 as a chromatographic system. Neutral Al 2 O 3 was used for column chromatographic separation. The products, after evaporation of the solvent, were purified by recrystallization from diethyl ether.
IR spectra were determined on a VERTEX 70 FT-IR spectrometer (Bruker Optics, Ettlingen, Germany). 1 H NMR and 13 C NMR spectra were recorded on a Bruker Avance III HD 500 spectrometer (Bruker, Billerica, MA, USA) at 500 MHz ( 1 H-NMR) and 125 MHz ( 13 C-NMR), respectively. Chemical shifts are given in relative ppm and were referenced to tetramethylsilane (TMS) (δ = 0.00 ppm) as an internal standard; the coupling constants are indicated in Hz. The NMR spectra were recorded at room temperature (ac. 295 K). HRESIMS spectra were acquired in positive mode on Q Exactive Plus (ThermoFisher Scientific, Inc., Bremen, Germany) mass spectrometer, equipped with a heated HESI-II source. Operating conditions for the HESI source used in a positive ionization mode were: +3.5 kV spray voltage, 320 • C capillary and probe heater temperature, sheath gas flow rate 36 a.u., auxiliary gas flow rate 11 a.u., spare gas flow rate 1 a.u. (a.u. refer to arbitrary values set by the Exactive Tune software) and S-Lens RF level 50.00. Nitrogen was used for sample nebulization and collision gas in the HCD cell. The aliquots of 1 µL of the solutions of the samples (ca. 20 µg mL −1 ) were introduced into the mass spectrometer through LC system Thermo Scientific Dionex Ultimate 3000 RSLC (Germering, Germany) consisting of 6-channel degasser SRD-3600, high-pressure gradient pump HPG-3400RS, autosampler WPS-3000TRS, and column compartment TCC-3000RS equipped with narrow bore Hypersil GOLD™ C18 (2.1 × 50 mm, 1.9 µm) column. Each chromatographic run was carried out isocratically with a mobile phase consisting of water-acetonitrile-methanolacetic acid (25:50:25:0.2). The solvent flow rate was 300 µL min −1 . Full MS-SIM was used as MS experiment in negative and positive mode, where resolution, automatic gain control (AGC) target, maximum injection time (IT), and mass range were 70,000 (at m/z 200), 3e6, 100 ms, and m/z 100-500, respectively. Xcalibur (Thermo Fisher Scientific, Waltham, MA, USA) ver. 4.0 was used for data acquisition and processing.

Synthetic Methods Experimental Protocols and Spectral Data
To a solution of 5 mmol of the starting ketone 3-methyl-1-phenylbutan-2-one 1 in 25 mL formamide, a catalytic amount of methanoic acid was added. The mixture was refluxed for 2 h at 180 • C, then poured in water and extracted with CH 2 Cl 2 (3 × 20 mL). The combined extracts were washed with Na 2 CO 3 solution, water, and dried using anhydrous Na 2 SO 4 , filtered on the short column filled with neutral Al 2 O 3, and then concentrated. The obtained formamide was directly hydrolyzed with 50 mL 5N H 2 SO 4 and 1 h reflux at 100 • C to 3-methyl-1-phenylbutan-2-amine 3. The mixture then was poured in water and extracted with CH 2 Cl 2 (2 × 20 mL). The water layer was alkalized with NH 4 OH and extracted with CH 2 Cl 2 (3 × 20 mL). The combined extracts were dried using anhydrous Na 2 SO 4 , filtered on the short column filled with basic Al 2 O 3, and then concentrated.

Theoretical Prediction of Pharmacokinetic Parameters (ADME)
Physicochemical properties, drug-likeness, and pharmacokinetic parameters such as ADME (absorption, distribution, metabolism, elimination) of the synthesized prodrugs were analyzed using SwissADME. It provides a predictive model for the pharmacokinetic profiling of a drug-like compound [22].

Theoretical Prediction of Toxicity
For predicting acute as well as organ toxicity of the compounds, the ProToxII web tool was used. It predicts various toxicity endpoints, including acute toxicity and organ toxicities such as hepatotoxicity, cytotoxicity, carcinogenicity, mutagenicity, immunotoxicity, and toxicity targets. Toxicity class and LD 50 values were also estimated [23,24].

PASS Online Predictions
PASS online (Prediction of Activity Spectra for Substances), a computer-based program, was used to screen biological activity of the compounds. The program predicts several thousand different biological activities based on the structural formula of a drug-like organic compound [25]. PASS has been used by many scientists for the discovery of new pharmaceutical agents in different therapeutic fields [26,27]. LBG agar was prepared by the manufacturer's (Laboratorios Conda S.A., Madrid, Spain) prescription: 50 g of LBG-solid substance mixture (containing 10 g tryptone, 5 g yeast extract, 10 g NaCl, 10 g glucose and 15 g agar) was dissolved in 1 L of deionized water (pH 7.5), and then the medium was autoclaved at 121 • C for 20 min.

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Malt extract agar (MEA) MEA was prepared by the manufacturer's (Laboratorios Conda S.A., Madrid, Spain) prescription: 50 g of the MEA-solid substance mixture (containing 30 g malt extract, 5 g mycological peptone, and 15 g agar) was dissolved in 1 L of deionized water (pH 5.4), and then the medium was autoclaved at 115 • C for 15 min.

Antimicrobial Activity Assay
The antimicrobial activity of the samples was determined by the agar well diffusion method. The tested bacteria B. subtilis and B. amyloliquefaciens were cultured on LBG agar at 30 • C. The test bacteria S. aureus, L. monocytogenes, E. faecalis, S. enteritidis, Klebsiella sp., E. coli, P. vulgaris and P. aeruginosa were cultured on LBG agar at 37 • C for 24 h. The yeast C. albicans was cultured on MEA at 37 • C, while S. cerevisiae was cultured on MEA at 30 • C for 24 h. The fungi A. niger, A. flavus, Penicillium sp., Rhizopus sp., Mucor sp., and F. moniliforme were grown on MEA at 30 • C for 7 days or until sporulation.
The inocula of the tested bacteria/yeasts were prepared by homogenization of a small amount of biomass in 5 mL of sterile 0.5% NaCl. The inocula of tested fungi were prepared by the addition of 5 mL of sterile 0.5% NaCl into the tubes. After stirring by vortex V-1 plus (Biosan), they were filtered and replaced in other tubes before use. The number of viable cells and fungal spores was determined using a bacterial counting chamber Thoma (Poly-Optik, GmbH, Bad Blankenburg, Germany). Their final concentrations were adjusted to 10 8 cfu/mL for bacterial/yeast cells and 10 5 cfu/mL for fungal spores and then inoculated in preliminarily melted and tempered at 45-48 • C LBG/MEA agar media. Next, the inoculated media were transferred in a quantity of 18 mL in sterile Petri plates (d = 90 mm) (Gosselin™, Hazebrouck, France) and allowed to harden. Then, six wells (d = 6 mm) per plate were cut, and triplicates of 60 µL of each extract were pipetted into the agar wells. The Petri plates were incubated in identical conditions.
The antimicrobial activity was determined by measuring the diameter of the inhibition zones around the wells on the 24th and 48th h of incubation. Tested microorganisms with inhibition zones of 18 mm or more were considered sensitive; moderately sensitive were those in which the zones were from 12 to 18 mm; resistant were those in which the inhibition zones were up to 12 mm or completely missing [28].

Cytotoxic Activity
In order to evaluate the in vitro biocompatibility of the experimental compounds, a series of cell viability assays were performed against human malignant leukemic cell lines (LAMA-84, K-562) as well as normal murine fibroblast cells (CCL-1). All cell lines were purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ GmbH, Braunschweig, Germany). Cell cultures were cultivated in a growth medium RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 5% L-glutamine, and incubated under standard conditions of 37 • C and 5% humidified CO 2 atmosphere.

Cell Viability Assay
The experimental design involved a number of cytotoxicity assays that measured cell growth inhibition by the newly synthesized compounds. Cell viability was evaluated using a standard MTT-based colorimetric assay. Exponential-phased cells were harvested and seeded (100 µL/well) in 96-well plates at the appropriate density (3 × 10 5 ) for the suspension cultures (LAMA-84 and K-562) and 1.5 × 10 5 for the adherent one (CCL-1). Cells were treated and incubated with various concentrations of the experimental compounds in the concentration range of 400-6.25 µM. After exposure time of 72 h, a filter-sterilized MTT substrate solution (5 mg/mL in PBS) was added to each well of the culture plate. A further 1-4 h incubation allowed for the formation of purple insoluble formazan precipitates. The latter were dissolved in isopropyl alcohol solution containing 5% formic acid prior to absorbance measurement at 550 nm using a microplate reader (Labexim LMR-1). Collected absorbance values were blanked against MTT and isopropanol solution and normalized to the mean value of the untreated control (100% cell viability). Semi-logarithmic "doseresponse" curves were constructed and the half-inhibitory concentrations of the screened compounds against each tested cell line were calculated. Values of p ≤ 0.05 were considered statistically significant. Adult male Wistar rats weighing 280-300 g were used. Animals were housed in standard laboratory conditions (23 to 25 • C, 50-55% humidity, 12/12 h light/dark cycle), while food and water were provided ad libitum.

Bioelectrical Activity
Circular SM preparations were dissected from the corpus gastric muscle (26-28 mm long and 0.8-0.9 mm wide; without mucosa). Two or three muscle strips were taken from the rat stomach. The number of SM preparations used for each data point is indicated by n. After dissection, the tissues must prevent loss of viability, so during the dissection, the tissues were systematically moistened with a solution (NaCl:KCl:CaCl 2 in ratio 27.2:1.1:1) prepared at 4 • C.
The BEA of SM preparations was investigated by means of the single sucrose gap method with silver non-polarizable freshly chlorinated electrodes. The muscle strips were divided into 3 zones: two electrode sections were flushed with isotonic KCl solution and Krebs solution (pH 7.3), respectively, and separated by a sucrose layer of high electric resistance. Krebs contained the following (in mM): NaCl 120; KCl 5.9; CaCl 2 2.5; MgCl 2 1.2; NaH 2 PO 4 1.2; NaHCO 3 15.4; glucose 11.5. All the ingredients for this solution were of analytical grade and obtained from Merck (Darmstadt, Germany). Deionized water (18.2 mX/cm 2 ) was used thoroughly. The KCl solution is similar to the Krebs solution but with an equimolar substitution of NaCl with KCl. An isotonic sucrose solution, due to its very high specific resistance (about 10 6 Ω·cm), provides the best possible separation of the sucrose solution from the physiological solution in the "normal" pool, on the one hand, and the solution providing a zero value of the membrane potential in the pool with another recording electrode.
The inflow rate of each solution (Krebs, KCl, and isotonic sucrose solution) was controlled by a needle valve and continuously aerated with a mixture of 95% O 2 and 5% CO 2 , and the temperature was maintained at 37 • C. A potential difference between the two end sections of the gap (control conditions) was achieved by means of silver nonpolarizable, freshly chlorinated electrodes and after a 40-min period of adaptation and stabilization was accepted as the initial (control). The Krebs solution was changed several times during the adaptation period. The viability of the SM preparations was periodically tested by treatment with 1 × 10 −6 M ACh.
The membrane potential value was visualized by means of a Linseis recorder (Selb, Germany). A pair of measuring electrodes is located on both sides of the sucrose sections. The pH (pH 7.3; 37 • C) of the Krebs bathing solution was measured before each experiment by pH-meter HI5521 (Hanna Instruments Inc., Woonsocket, RI, USA).
The CAs were recorded from the fragments of the SM preparations located in the section with Krebs solution. The mechanical changes were registered isometrically with a Microtechna amplifier (Prague, Czech Republic) and recorded by a Linseisrecorder (Selb, Germany).
The new substances were dissolved initially in dimethyl sulphoxide (DMSO) to prepare a 5 × 10 −2 M stock solution. Further dilutions were made in distilled deionized water and were applied by adding aliquots of their concentrated solutions to the tissue bath. The final concentration of the studied substances in the tissue baths did not exceed 1:100.

Histology
After dissection, the SM strips were incubated for 20 min in the tissue bath using the same physiological conditions in which the CA recording experiments were carried out. Then, each SM strip was fixed in 10% neutral buffer formalin for 24-48 h and submitted for standard processing with hematoxylin and eosin staining.
The SM fragments from a rat's gastric wall were fixed in a 10% neutral formalin solution and embedded in paraffin. Paraffin sections of 5 µm thickness were subjected to hematoxylin and eosin (H-E) staining for histochemical analysis.

Hematoxylin-Eosin Staining
The sections were stained with Mayer's hematoxylin (5 min) and differentiated in tap water (20 min). After completion of the differentiation, the sections were stained with water-soluble eosin for 5 min. The subsequent stained sections were dehydrated again in ascending grades of ethanol (70%, 95%, 100%), cleared in xylene (2 × 10 min), and covered with Canadian balm.

Immunohistochemistry
Sections of the SM fragments from the wall of a rat's stomach with a thickness of 5 µm were deparaffinized, then subjected to antigenic detection of the epitopes with citrate buffer, and an endogenous peroxidase blockade was made with hydrogen peroxidase 3%, a kit was used to block the endogenous biotin, and a reagent was used to block non-specific binding (Superblock, Scy Tek, Santa Cruz Biotech. Inc. Heidelberg, Germany) follows incubate for 24 h (at 4 • C) with monoclonal mouse anti-5-HT 3 , next incubated with a secondary antibody: biotinylated anti-rabbit for 10 min at room temperature. The reaction was visualized with 3,3 -diaminobenzidine tetrachloride and counterstained with Mayer's hematoxylin. The preparations were observed with a light microscope at magnification levels of ×200.

Quantitative Analysis of Immunohistochemical Reactions
A quantitative and statistical analysis of immunohistochemical reaction using the Olympus DP-Soft image system (version 4.1 for Windows) was carried out on a Microphot-SA (Nikon, Japan) microscope equipped with a Camedia-5050 Z digital camera (Olympus, Japan). The analysis was performed on sections from the SM strips from the stomach of Wistar rats (n = 6 for each group). Five sections of the SM strips were measured, and the percentage of cells expressing 5-HT 3 in the circular and longitudinal layer of SM cells, as well as in the myenteric plexus of the stomach, was determined. Each antibody was analyzed for five fields, in each of them the average number of cells with positive unit area response at ×200 magnification.

Statistical Analysis
All data were expressed as mean values ± standard error of the mean (mean ± SEM); the number of muscle preparations used for each data point was indicated by n. The results were analyzed by analysis of variance (ANOVA) to find the values that showed significant difference (p < 0.05). INSTAT computer program (GraphPad Software, Inc., San Diego, CA, USA) was applied for the analysis of experimental data.

Results and Discussion
Investigating the biological activity of the newly synthesized compounds is a relevant scientific matter due to the interest in the drug discovery area. In our previous reports, we discussed the synthesis and SM BEA for different compounds, such as 1,2,3,4tetrahydroisoquinolines [29], isoquinoline precursors [6], melanoidins [30,31], eucalyptol, thymol [32,33], etc.

In Silico Predictions and Synthesis
To be effective as a drug, a given molecule must reach its pharmacological target in the body, achieve an adequate concentration at the site of action, and persist in a bioactive form long enough for the expected biological events to occur. Many compounds failed as drugs because of their poor pharmacokinetics properties and limited bioavailability. The large number of newly synthesized experimental molecules, on one hand, and the quantitative limitations of tissue samples, together with the need to restrict animal testing, on the other, prevent systematic recourse to experiments. In this context, in silico computer models constitute a valid alternative and a useful addition to biological experiments [34]. In our calculations, the PASS Online Program (Prediction of Activity Spectra for Substances) was used. The latter predicted the potential spasmolytic activity of the tested compounds based on a papaverine-like structure, and their modulatory effect on serotonin kinetics (release and uptake of the mediator) and its receptor transmission.
Our strategy was based on the synthesis of 3-methyl-1-phenylbutan-2-amine from a starting ketone and its acylation with acid halides to prepare corresponding amides. To obtain the compounds, we successfully used a previously described protocol [6] using 3-methyl-1-phenylbutan-2-one 1 as a starting compound. The ketone applied in Leuckart reaction with HCOOH and HCONH 2 and reflux for 2 h at 180 • C produced formamide 2 which after hydrolysis in 5N H 2 SO 4 obtained the target amine 3 with 80% yield, according to the following Scheme 1. Due to the importance of their synthesis as isoquinoline precursors and antispasmodics, a target amine 3 and its new amides were successfully achieved in order to investigate their biological activity. The synthesis of amides is extremely important due to the high presence of this group in biological systems, as well as its key role in medicinal chemistry, where one of the most common chemical reactions is amide synthesis. Amide groups are also synthetically versatile, capable of taking part in a wide range of different transfor- Due to the importance of their synthesis as isoquinoline precursors and antispasmodics, a target amine 3 and its new amides were successfully achieved in order to investigate their biological activity. The synthesis of amides is extremely important due to the high presence of this group in biological systems, as well as its key role in medicinal chemistry, where one of the most common chemical reactions is amide synthesis. Amide groups are also synthetically versatile, capable of taking part in a wide range of different transformations. It is difficult to overstate the importance of the amide functional group in molecular sciences. This motif is of crucial importance to form the backbone of peptides, proteins, and other biomolecules [35]; the amide-forming reactions being the most commonly carried out in the pharmaceutical industry [36]. Approximately a quarter of all marketed drugs and many of all drug candidates bear an amide bond [37]. Amide interactions with biological targets are another key aspect to be considered in drug discovery [38].
Based on the in silico results, we worked on a synthetic route to apply different acyl chlorides, bearing electron-donating and electron-withdrawing groups as the starting material (Scheme 2, Table 1).
Due to the importance of their synthesis as isoquinoline precursors and antispasmod ics, a target amine 3 and its new amides were successfully achieved in order to investigat their biological activity. The synthesis of amides is extremely important due to the high presence of this group in biological systems, as well as its key role in medicinal chemistry where one of the most common chemical reactions is amide synthesis. Amide groups ar also synthetically versatile, capable of taking part in a wide range of different transfor mations. It is difficult to overstate the importance of the amide functional group in molec ular sciences. This motif is of crucial importance to form the backbone of peptides, pro teins, and other biomolecules [35]; the amide-forming reactions being the most commonly carried out in the pharmaceutical industry [36]. Approximately a quarter of all marketed drugs and many of all drug candidates bear an amide bond [37]. Amide interactions with biological targets are another key aspect to be considered in drug discovery [38].
Based on the in silico results, we worked on a synthetic route to apply different acy chlorides, bearing electron-donating and electron-withdrawing groups as the starting ma terial (Scheme 2, Table 1). In general, reactions with different acyl chlorides proceeded efficiently, furnishing the target amides 4a-d in 69-83% yield (Table 1). Comparing the yield with previously reported compounds having a 3-methyl group [6], we can conclude that the yield for 3 isopropyl substituted compounds has decreased because of the lack of methoxy group in the benzene ring. The resultant compounds are characterized by their melting point IR, 1 H, 13 C-NMR, and HRESIMS spectra. Spectral data confirmed the structure of all th obtained compounds (Supplementary Materials Figures S1-S20).  In general, reactions with different acyl chlorides proceeded efficiently, furnishing the target amides 4a-d in 69-83% yield (Table 1). Comparing the yield with previously reported compounds having a 3-methyl group [6], we can conclude that the yield for 3-isopropyl substituted compounds has decreased because of the lack of methoxy groups in the benzene ring. The resultant compounds are characterized by their melting point, IR, 1 H, 13 C-NMR, and HRESIMS spectra. Spectral data confirmed the structure of all the obtained compounds (Supplementary Materials Figures S1-S20).

Scheme 2. Synthesis of N-(1-(3,4-dimethoxyphenyl)propan-2-yl) amides 4.
The blood-brain barrier (BBB) and gastrointestinal absorption are pharmacokinetics characteristics that play an essential role in the drug discovery process. In order to elucidate the ADME profile of the compounds, free SwissADME web tools were used ( Table 2).
Molecular weight (MW) and octanol/water partition coefficient (XLOGP3) are the drug-likeliness parameters, known as Lipinski's rule of five [39]. According to the rule, at least two parameters from four basic pharmacokinetic properties (MW ≤ 500; XLOGP3 ≤ 5; the number of hydrogen bond donors ≤ 5; and hydrogen bond acceptors ≤ 10.6) should be fulfilled for drug candidates. Bioavailability (BA) [40] reported an optimal range of distinct properties involving lipophilicity (XLOGP3: −0.7 to +5.0), size (MW: 150 to 500 g/mole), polarity (TPSA: 20 to 130 Å 2 ), ESOL or estimated solubility (log S: not more than 6), saturation (Fraction Csp 3 or fraction of carbons in the sp 3 hybridization: not less than 0.25), and flexibility (RB: no more than 9). The ADME investigation shows that the compounds cross through the BBB and present good gastrointestinal absorption and could be considered as a drug candidate according to Lipinski's rule of five [39], an important step in a drug discovery process. The calculated TPSA values of the compounds are in the range of 26.02-29.10 Å 2 , indicating good intestinal absorption and BBB penetration. The low number of rotatable bonds (3, Table 2) for 3 and 4a compounds corresponds with sufficient oral bioavailability. P-glycoproteins are pharmacokinetic proteinsfunctioning as xenobiotic neuroprotectors, drugs, etc. [41,42] According to the calculation, the studied compounds are not predicted as P-glycoprotein substrates. It is known that inhibition of cytochrome P450 (CYP) could lead to toxicity or lack of drug efficacy [43,44]. According to the calculations, all the compounds are expected to inhibit the CYP2D6 isoform. Compounds 4c and 4d with a phenyl ring can inhibit the CYP1A2 and CYP2C19, while chlorine containing 4d-the CYP2C9. Compounds 3 and 4a have the lowest skin permeability with log Kp −5.46 and −5.50. The log Kp coefficients of 4b and 4c are between −4.46 and −4.83.
The solubility parameter is very important for the pharmaceutical applications of the drug. All drugs were predicted by SwissADME to be water-soluble or moderately soluble in water. The values of the drug-likeness parameters of all compounds were found to remain within the BA, which confirmed that all the synthesized compounds behave as "drug-like" substances.
All of the compounds were calculated to have a BA score of 0.55, which demonstrates good oral bioavailability, according to Lipinski's rule [39]. Moreover, they have good synthetic accessibility (SA) scores, which are considerable parameters during the drug discovery processes. The predicted LD 50 for all the compounds is between 241 mg/kg and 2500 mg/kg.
The newly synthesized compounds were then subjected to an in vitro evaluation of their antimicrobial and cytotoxic activity against human malignant leukemic cell lines (LAMA-84, K-562) as well as normal murine fibroblast cells (CCL-1).

Antimicrobial Activity
Biofilms are aggregated bacteria attached to surfaces and embedded in a self-produced matrix of extracellular polymeric substances. The local environment within a biofilm offers protection to persister cells from the immune system [45]. These bacterial biofilms can be formed on any surface, including medical implants or water distribution systems in hospitals [46]. Bacteria, especially in biofilm, can be extremely difficult to eradicate and cause therapeutic problems, as is the case with the critical pathogen P. aeruginosa. Antibiotic resistance is one of the most persistent issues worldwide nowadays and many powerful antibiotics have failed in controlling infections. Recently, Krishnan and Kandasamy investigated the possibility of using the antispasmodic mebeverine against methicillin-resistant Staphylococcus aureus. They proved mebeverine to be favorable in the antibiotics dose reduction, and cutting down the need for additional administration of antibiotics to the patients affected with multiple complications such as gastrointestinal ulcer, spasm difficulties, and infection [47][48][49].
In our experiments, five Gram-positive bacteria, five Gram-negative bacteria, two yeasts, and six fungi were used. All the mebeverine precursors were tested in vitro for their antimicrobial activity against human pathogenic bacteria and economically relevant phytopathogenic fungi. The inhibition zones of bacterial and fungal growth caused by the novel compounds are outlined in Table 3. The methanol used as a solvent for the samples did not show any antimicrobial effect. Table 3. Antimicrobial activity of the tested compounds. 3  4a  4b  4c  4d Mebeverine Methanol

Inhibition Zones, mm Tested Microorganism/Compound
We observed that the amine 3 derivative exerted only modest activity against Gramnegative bacteria including the most pathogenic Klebsiella sp., Pseudomonas aeruginosa, as well as against Saccharomyces cerevisiae. The amide 4a, on the other hand, inhibited only the growth of Escherichia coli. None of the other amides exhibited any antimicrobial activity. This trend led us to the conclusion that antibacterial properties are distinctive to the amine and less substituted amides, such as acetyl, whereas amides with bulkier phenyls or benzyl group substituents did not show any antimicrobial activity. The most important conclusion in our experiments is that mebeverine did not show any antimicrobial activity in the same concentration range. This gives us an opportunity to further investigate biological activities of the synthesized more potent mebeverine precursors.

Cytotoxic Activity
A series of MTT experiments were conducted against normal murine fibroblast cells and malignant human cell lines of leukemic origin to accommodate the cytotoxicity of the target compounds. The results of the antiproliferative assays are presented in Table 4. According to our study, cell growth was unaffected following 72 h exposure to various treatment concentrations of the compounds in all the screened cell lines. Calculated IC 50 values range in the 240-360 µM for the leukemic models and lie strictly above 300 µM in the normal fibroblast cells. The compounds induced no morphological changes under microscopic examination and were practically devoid of any intrinsic cytotoxic activity in the tested treatment concentrations that many times exceed their spasmolytic (5 × 10 −5 M) dose. Based on the PASS online and the ProToxII in silico calculations, the evaluation of the cytotoxic activity of the compounds was the other object of our investigation. We found that the compounds induced no morphological changes under microscopic examination and were practically devoid of any intrinsic cytotoxic activity in the tested treatment concentrations. Furthermore, they showed a more favourable cytotoxicity profile as compared to the spasmolytic drug used as reference (mebevirine), whose half-inhibitory concentrations on the malignant cell lines LAMA-84 and K-562 fall just under the hundred-micromolar range (Table 4).

Ex Vivo Effects of 3-Methyl-1-phenylbutan-2-amine 3 and Its Amides 4a-d on the Spontaneous Bioelectric Activity
Taking into account the potential effect of the compounds on muscle tonus, the spontaneous BEA on SM isolated from the rat stomach was established. The response of a living cell to various stimuli, whether of an excitation origin producing a bioelectrical response or of a chemical nature, always occurs after some latent lag period. This time interval (meantime) is relatively short in the cells of nerve and muscle tissue, which are adapted to carry out rapid responses to irritation. In SMs, the excitation process is associated with changes in BEA, because the sarcolemma of most of them has potential-dependent ion channels and, for this reason, belongs to the electro-excitable membranes. Gastric SMs are spontaneously active and generate rhythmic activities such as slow waves or action potentials, or both [50]. The membrane potential of SM cells has a relatively low value of −30 mV to −65 mV. Its absolute value with a suprathreshold electrical or chemical stimulus reaches the threshold of the opening of the potential-dependent Ca 2+ channels and the entry of calcium into the sarcolemma [51]. The increased amount of Ca 2+ in the cytosol causes the generation of the depolarization phase of the spike potentials in the cells, which cause phasic contractions of the muscle cells [50]. When intracellular recordings are made from the SM preparation, three types of electrical responses are found: pacemaker potentials, slow waves, and follower potentials [52]. In our experiments, the mean response time of SM cells was in the order of 10 min for the complete change in slow wave character to develop. Membrane potential value was significantly increased by 55% after the addition of 3 into the tissue bath. That probably depends on the activation of voltage-gated Ca 2+ ion channels in which the Ca 2+ influx is observed.
Affecting this functionally important process is also confirmed by the significant increase in the frequency of spike potentials ( Figure 4; Table 5), responsible for SM contractions [53]. Table 5. Changes of some bioelectric parameters of SM preparations registered through sucrose gap method after influence of the newly synthesized mebeverine precursors 5 × 10 −5 M (n = 7). The asterisk indicates the values that differ significantly (* p < 0.05) compared to the respective control parameters; base activity is taken as 100%, ↑ show depolarization; ↓-hyperpolarization. Relaxation of smooth muscle occurs by two mechanisms: a hyperpolarization of the membrane through activation of K ATP channels and elevation of intracellular cGMP levels due to its role as a nitric oxide (NO) donor [54]. Hyperpolarization of the membrane has two distinct actions on slow waves: reduction in the amplitude and a shift in the threshold potential to more negative levels [55,56].

Immunoreactivity for the 5-HT3 Receptor
Finally, a semiquantitative analysis of the immunohistological appearance o preparations incubated with 5-HT and the newly synthesized substances was pe ( Figure 5). Analysis of the results of 5-HT3 receptors expression in gastric SM showed a higher number of 5-HT3R positive cells per section when treated w pound 3 or 5-HT separately. In the preparations incubated with the newly syn compounds 4a, 4b, and 4c, no modulation on the 5-HT3R expression was observe arations incubated with compound 4d showed a very weak non-specific effect on otoninergic 5-HT3R. Our experiments with single sucrose gap method [57] showed a reduction in the frequency and amplitude of spike potentials generated by SM preparations in the presence of the amide 4d ( Figure 4E). Spike potentials are known to be a product of Ca 2+ currents through L-type membrane Ca 2+ channels and the significant reduction in their indicators under the influence of 4d is an indication of the probable participation of this type of membrane ion channels in the bioelectric activity of the studied substances.
We found that exogenous administration of 4a unequivocally demonstrated increased reactivity in SM rat stomach preparations. We registered a complete disappearance of the spike potentials responsible for SM contractility. The biological activity of amide 4a is also confirmed by hyper-polarization, expressed in a reliable shift of the value of the membrane potential to more negative values ( Figure 4B).
The BEA of SM preparations for substances 4b and 4c was also investigated. The data do not demonstrate any changes in their slow waves ( Figure 4C,D). Based on the experiments, we can assume that 4b and 4c do not affect the BEA of the isolated tissues studied by the single sucrose gap method.
We also accepted the possibility of modulating the general bioelectrical mechanisms inducing contractility in SM. We assumed that the different nature of the responses to exogenously applied compounds 3, 4a, and 4d as new molecules in their capacity as excitatory substances is due to the specificity in the type of the substituents and their position. In order to analyze the validity of this hypothesis, measurements of possible changes in the BEA of SM preparations for substances 4b and 4c were made. The data do not demonstrate any changes in their slow waves. Therefore, we can assume that 4b and 4c do not affect the BEA of the isolated tissues studied by the single sucrose gap method.
Which one of the mentioned processes and how is affected by the newly synthesized substances are our next goals to be investigated by tracking isometric changes in the contractile activity of SM preparations, based on the already proven influence on the bioelectrical changes that initiate them.

Immunoreactivity for the 5-HT 3 Receptor
Finally, a semiquantitative analysis of the immunohistological appearance of the SM preparations incubated with 5-HT and the newly synthesized substances was performed ( Figure 5). Analysis of the results of 5-HT 3 receptors expression in gastric SM strips showed a higher number of 5-HT 3 R positive cells per section when treated with compound 3 or 5-HT separately. In the preparations incubated with the newly synthesized compounds 4a, 4b, and 4c, no modulation on the 5-HT 3 R expression was observed. Preparations incubated with compound 4d showed a very weak non-specific effect on the serotoninergic 5-HT 3 R.
The results of the immunohistological analysis showed that amine 3 has the ability to induce the expression of 5-HT 3 receptors on afferent neurons and nerve fibers in the Myenteric plexus of SM preparations ( Table 6). Administration of 3 activates 5-HT 3 receptors, possibly causing rapid inward currents (or membrane depolarization) in neurons consistent with 5-HT 3 R function as a ligand-gated cation channel. This suggests that amine 3 has the potential to modulate afferent neuronal activity at distinct sites of the gastrointestinal tract and may therefore prolong or enhance local GIT signaling. Table 6. 5-HT expression and response to compounds 3, 4a-d compared to mebeverine hydrochloride (MH).

Conclusions
In conclusion, a new amine and a series of its amide derivatives were synthesized, as mebeverine precursors and antispasmodics. Generally, all the synthesized compounds showed favourable predicted in silico profiles against the majority of the selected descriptors. In silico data signified amides as potential orally active medicinal substances with reduced toxicity. Only amine 3 demonstrated modest antimicrobial activity against Gram-negative bacteria Klebsiella sp., Pseudomonas aeruginosa, and yeasts Saccharomyces cerevisiae. We can conclude that the antimicrobial activity of the amine was higher than the mebeverine activity in the same concentration range. Overall, no cytotoxicity to human malignant leukemic cell lines (LAMA-84, K-562) was observed for all tested compounds. We established ex vivo BEA using functionally active isolated tissues. The results showed that three of the synthesized substances 3, 4a, and 4d exhibit BEA, expressed in a reliable change of the main characteristics of the slow wave-the value of the membrane potential, the frequency, and amplitude of the wave, and the spike potentials. Furthermore, the immunohistochemical analysis indicated that amine 3 has the potential to modulate afferent neuronal activity at distinct sites of the gastrointestinal tract and may therefore prolong or enhance local gastrointestinal signaling.
The summary results of the conducted experiments distinguished the newly synthesized 3-methyl-1-phenylbutan-2-amine 3 as a leader compound with favourable antimicrobial, immunohistochemical and spasmolytic activity, and a reasonable effective choice for orally active long-term therapy of chronic IBS. Thus, further investigation into the possible medicinal applications of this compound is warranted in the future.